Glucose-induced insulin secretion from pancreatic islets requires metabolism of glucose within islet b-cells, and ATP has attracted interest as a messenger of glucose metabolism within a-cells. Glucose-induced Insulin secretion from islets and HIT insulinoma cells Is accompanied by activation of an ATP-stimulatable Ca2+-Independent PLA2 (ASCl-PLA2) enzyme, the catalytic activity of which resides in a 40 kDa protein [Ramanadham et al., 1993a, 1994]. An analogous Pl-A2 enzyme in myocardium was recently demonstrated to consist of a complex of a 40 kDa catalytic protein with a tetramer of an iso-form of the glycolytic enzyme phosphofructokinase (PFK) [Hazen & Gross, 1993]. Association of the PFK-isoform with the myocardial PLA2 catalytic protein was found to confer ATP-sensitivity to the enzyme complex. Here we demonstrate that the majority of HIT cell and islet ASCI-PLA2 catalytic activity elutes from a gel filtration column in a region corresponding to 400 kDa, suggesting that the 40 kDa B-cell ASCI-PLA2 catalytic protein exists as part of a larger molecular weight complex. Islet and HIT cell ASCI-PLA2 activities were Immunoprecipitated by antibodies directed against PFK, and the immunoprecipitates contained 40 kDa and 85 kDa proteins which correspond to the molecular masses of the PLA2 catalytic protein and of PFK, respectively. Islet and HIT cell ASCI-PLA2 activities were selectively arid reversibly adsorbed to affinity matrices containing immobilized PFK but not to similar matrices containing Immobilized transferrin or bovine serum albumin. Addition of free PFK prevented binding of HIT cell ASCI-PLA2 activity to immobilized PFK-matrices and promoted desorption of activity previously bound to sich matrices. These results suggest that B-cell ASCI-PLA2, like the myocardial enzyme, exists as a complex comprised of a catalytic protein and a PFK-like protein and raise the possibility that the ASCI-PLA2 complex may represent a component of the A-cell glucose-sensor, which links glycolysis, phospholipid hydrolysis, and membrane electrochemical events involved In glucose-Induced insulin secretion.